Transcription Factor Gene Osnacx From Rice and Use Thereof for Improving Plant Tolerance to Drought and Salt

ABSTRACT

The present invention relates to an isolated polynucleotide capable of giving a plant tolerance to drought and/or salt stress, which comprises a polynucleotide sequence as shown in SEQ ID NO:1, and to a promoter capable of giving a plant tolerance to drought and/or salt stress. The present invention also relates to an expression vector comprising the said polynucleotide and/or the said promoter, and to a host cell transformed or transfected by the said expression vector. The present invention further relates to a use of the said polynucleotide or promoter sequence in improvement of plant tolerance to drought and/or salt stress.

TECHNICAL FIELD

The present invention relates to an isolated polynucleotide capable ofgiving a plant tolerance to drought and/or salt stress, which comprisesa polynucleotide sequence as shown in SEQ ID NO:1, and to a promotercapable of giving a plant tolerance to drought and/or salt stress. Thepresent invention also relates to an expression vector comprising thesaid polynucleotide and/or the said promoter, and to a host celltransformed or transfected by the said expression vector. The presentinvention further relates to a use of the said polynucleotide orpromoter sequence in improvement of plant tolerance to drought and/orsalt stress.

BACKGROUND ART

The growth of plants usually are influenced by many environmentalfactors, wherein drought and/or salt damage are main factors resultingin great reduction of crop production in many areas. Thus, it always amajor aim to develop crop species with stress tolerance in researches ofagricultural science and technology.

For resisting or adapting to disadvantageous environmental factors,plants receive extracellular changes of environmental conditions andtransfer them through many pathways into cells to induce expressions ofsome responding genes and generate some functional proteins,osmoregulation substances as well as transcription factors for signaltransmission and gene expression regulation so that plants are able tomake corresponding responses to environmental changes and avoid damagescaused by drought, high salt and/or low temperature stresses. (Xiong etal, Cell signaling during cold, drought and salt stress. Plant Cell. 14(suppl), S165-S183, 2002). The regulating factors finely regulate theexpression of functional genes for responding environmental changes.When plants encounter stresses, transcription factor as a controllinggene is able to regulate the expression of a series of downstream genesto enhance the tolerance of plants to the stresses.

Kawasaki et al (2001) utilized microarrays to analyze the earlyexpression profile of rice under high salt stress, and disclosed that agreat number of genes were induced or inhibited and the induction andexpression of these genes were regulated by transcription factor(Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K,Galbraith D and Bohnert H J. Gene expression profiles during the initialphase of salt stress in rice, Plant Cell, 2001, 13: 889-905). It isfound that transcription factor families of AP2/EREBP, Zinc finger, Myb,bZIP in Arabidopsis thaliana are induced to be expressed or inhibitedunder different stresses (Shinozaki K et al, Monitoring the ExpressionPattern of 1300 Arabidopsis Genes under Drought and Cold Stresses byUsing a Full-Length cDNA Microarray, Plant Cell, 2001, 13: 61-72). Thus,it is deemed that these transcription families are very important inregulation during the procedure of plant response to stresses.Therefore, the separation and identification of transcription factorshaving core regulation function for response to stresses and the usethereof for genetic improvement of crop to resist stresses are importantand meaningful for seed breeding.

Based on the known information of Arabidopsis thaliana transcriptionfactors, some studies have been done to improve plant tolerances.Transgenic Arabidopsis thaliana plants cultured by using EREB1A andDREB2A have higher tolerances to low temperature, drought and highsalinitiy than the wild type (Liu Q et al, Two transcription factors,DREB1 and DREB2, with an EREBP/AP2 DNA domains separate two cellularsignal thansduction pathways in drought—and low-temperature-responsivegene expression, respectively, in Arabidopsis. Plant Cell. 1998, 10:1391-1406). The research group of Thomashow M F in Michigan StateUniversity (U.S.A.) also cultured plants with enhanced freezingtolerance by using Arabidopsis thaliana CBF1 gene in genetictransformation.

Rice is one of the most important alimentary corps. The tolerance todrought and/or salt is particularly important for rice. However, notransgenic rice plant with tolerance to drought and/or salt has beendeveloped so far. Thus, it is meaningful and important to find outtranscription factor associated with tolerance to drought and/or saltfor culturing a rice plant with tolerance to drought and/or freezing andthereby increasing rich production.

SUMMARY OF THE INVENTION

One object of the present invention is to provide an isolatedpolynucleotide capable of giving a plant, preferably rice, tolerance todrought and/or salt stress, which comprises a nucleotide sequence asshown in SEQ ID NO:1, or a conservative variant or degenerate sequencecomprising one or more substitutions, deletions, additions and/orinsertions in the said nucleotide sequence, or a sequence hybridizablewith the said sequence under moderate stringent condition, or acomplementary sequence thereof, or a variant or derivative having atleast 95% homology and same or similar biological function to the saidnucleotide sequence.

In one embodiment of the present invention, the said polynucleotideconsists of the DNA sequence as shown in SEQ ID NO:1. In anotherembodiment of the present invention, the said polynucleotide consists ofthe DNA sequence as shown in the positions 1374-2453 of SEQ ID NO:1.

Another object of the present invention is to provide a promoter capableof giving a plant, preferably rice, tolerance to drought and saltstress, which comprises a nucleotide sequence as shown in the DNAsequence of the positions 1-1373 of SEQ ID NO:1, or a conservativevariant or degenerate sequence comprising one or more substitutions,deletions, additions and/or insertions into the said nucleotidesequence, or a sequence hybridizable with the said sequence undermoderate stringent condition, or a complementary sequence thereof, or avariant or derivative having at least 95% homology and same or similarbiological function to the said nucleotide sequence. In one embodimentof the present invention, the said promoter consists of the DNA sequenceas shown in the DNA sequence of the positions 1-1373 of SEQ ID NO:1.

Another object of the present invention is to provide an expressionvector comprising the said polynucleotide sequence and/or the saidpromoter sequence.

Another object of the present invention is to provide a host celltransformed or transfected by the said expression vector.

Another object of the present invention is to provide a use of the saidpolynucleotide sequence and/or the said promoter for increasingtolerance to drought and/or salt stress in plant, preferably rice.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is to isolate a DNA fragmentcomprising transcription factor gene complete encoding region, to cloneit, and to use it for improvement of tolerance of rice or other plantsto drought. The present invention is based on the discovery by structureanalysis of the obtained gene that belongs to plant-specifictranscription factor NAC family, and thus the said transcription factoris named as OsNACx.

In the present invention, the term “isolated polynucleotide capable ofgiving a plant tolerance to drought and/or salt stress” represents thepolynucleotide sequence as shown in SEQ ID NO:1, and further comprisesall variants or derivatives having at least 95% homology and same orsimilar biological function to the sequence as shown in SEQ ID NO:1.

The term “isolated” means “artificially” changed from natural statusand/or isolated from natural environment. Thus, if an “isolated”component or substance existing in nature is “isolated”, it has beenchanged or removed from its initial environment or been subject to both.For example, a polynucleotide or polypeptide naturally existing in liveanimal is not “isolated”, but the same polynucleotide or polypeptideisolated from its natural status is “isolated”, which is exactly theterm used herein.

The term “polynucleotide(s)”, as used herein, means a single or doublestranded polymer of deoxyribonucleotide or ribonucleotide bases andincludes DNA and corresponding RNA molecules, including HnRNA and mRNAmolecules, both sense and anti-sense strands, and comprehends cDNA,genomic DNA and recombinant DNA, as well as wholly or partiallysynthesized polynucleotides. An HnRNA molecule contains introns andcorresponds to a DNA molecule in a generally one-to-one manner. An mRNAmolecule corresponds to an HnRNA and DNA molecule from which the intronshave been excised. A polynucleotide may consist of an entire gene, orany portion thereof. Operable anti-sense polynucleotides may comprise afragment of the corresponding polynucleotide, and the definition of“polynucleotide” therefore includes all such operable anti-sensefragments.

A nucleotide “variant” is a sequence that differs from the recitednucleotide sequence in having one or more nucleotide deletions,substitutions or additions. Such modifications may be readily introducedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis as taught, for example, by Adelman et al.(DNA, 2: 183, 1983). Nucleotide variants may be naturally occurringallelic variants, or non-naturally occurring variants. Variantnucleotide sequences preferably exhibit at least about 70%, morepreferably at least about 80% and most preferably at least about 90%homology (determined as described below) to the recited sequence.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology or completehomology (in other words, identity). “Sequence identity” refers to ameasure of relatedness between two or more nucleic acids, and is givenas a percentage with reference to the total comparison length. Theidentity calculation takes into account those nucleotide residues thatare identical and in the same relative positions in their respectivelarger sequences. Calculations of identity may be performed byalgorithms contained within computer programs such as “GAP” (GeneticsComputer Group, Madison, Wis.) and “ALIGN” (DNAStar, Madison, Wis.). Apartially complementary sequence is one that at least partially inhibits(or competes with) a completely complementary sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous”. The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (in other words, the hybridization) of a sequence which iscompletely homologous to a target under conditions of low stringency.This is not to say that conditions of low stringency are such thatnon-specific binding is permitted; low stringency conditions requirethat the binding of two sequences to one another be a specific (in otherwords, selective) interaction. The absence of non-specific binding maybe tested by the use of a second target which lacks even a partialdegree of complementarity (for example, less than about 30% identity);in the absence of non-specific binding the probe will not hybridize tothe second non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas acDNA or genomic clone, the term “substantially homologous” refers toany probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas describedin.

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄×H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5g Ficoll (Type400, Pharmacia), 5g BSA (Fraction V; Sigma)] and 100 μg/ml denaturedsalmon sperm DNA followed by washing in a solution comprising 5×SSPE,0.1% SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄×H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100: g/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

It is well known that numerous equivalent conditions may be employed tocomprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (forexample, the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions that promote hybridization underconditions of high stringency (for example, increasing the temperatureof the hybridization and/or wash steps, the use of formamide in thehybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described above.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(in other words, it is the complement of) the single-stranded nucleicacid sequence under conditions of low to high stringency as describedabove.

The term “hybridization” refers to the pairing of complementary nucleicacids. Hybridization and the strength of hybridization (in other words,the strength of the association between the nucleic acids) is impactedby such factors as the degree of complementary between the nucleicacids, stringency of the conditions involved, the T_(m) of the formedhybrid, and the G:C ratio within the nucleic acids. A single moleculethat contains pairing of complementary nucleic acids within itsstructure is said to be “self-hybridized”.

The term “T_(m)” refers to the “melting temperature” of a nucleic acid.The melting temperature is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half dissociated intosingle strands. The equation for calculating the T_(m) of nucleic acidsis well known in the art. As indicated by standard references, a simpleestimate of the T_(m) value may be calculated by the equation:T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at1M NaCl (See for example, Anderson and Young, Quantitative FilterHybridization (1985) in Nucleic Acid Hybridization). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” refers to the conditions oftemperature, ionic strength, and the presence of other compounds such asorganic solvents, under which nucleic acid hybridizations are conducted.With “high stringency” conditions, nucleic acid base pairing will occuronly between nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “low” stringency areoften required with nucleic acids that are derived from organisms thatare genetically diverse, as the frequency of complementary sequences isusually less.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e. the window size) and multiplying the results by 100 toyield the percentage of sequence identity. In general, all or a portionof polynucleotides described herein may be prepared using any of severaltechniques.

In other words, for obtaining a polynucleotide with a nucleotidesequence having at least 95% identity to the reference nucleotidesequence, up to 5% nucleotides in the reference sequence could bedeleted or substituted by other nucleotides; or up to 5% nucleotideswith reference to the total nucleotides of the reference sequence couldbe inserted into the reference sequence; or up to 5% nucleotides withreference to the total nucleotides of the reference sequence could besubject to a combination of deletion, insertion and substitution. Thesemutations in the reference sequence could occur at 5- or 3-terminalposition of the reference nucleotide sequence or at any position betweenthese terminal positions, and they exist in the reference nucleotidesequence either in individual manner or in one or more adjacent groups.

One aspect of the present invention relates to an isolatedpolynucleotide capable of giving a plant tolerance to drought and/orsalt stress, which comprises a nucleotide sequence as shown in SEQ IDNO:1, or a conservative variant or degenerate sequence comprising one ormore substitutions, deletions, additions and/or insertions into the saidnucleotide sequence, or a sequence hybridizable with the said sequenceunder moderate stringent condition, or a complementary sequence thereof,or a variant or derivative having at least 95% homology and same orsimilar biological function to the said nucleotide sequence.

In one embodiment of the present invention, the said polynucleotideconsists of the DNA sequence as shown in SEQ ID NO:1. In anotherembodiment of the present invention, the said polynucleotide consists ofthe DNA sequence as shown in the positions 1374-2453 of SEQ ID NO:1.

Another aspect of the present invention relates to a promoter capable ofgiving a plant, preferably rice, tolerance to drought and salt stress,which comprises a nucleotide sequence as shown in the DNA sequence ofthe positions 1-1373 of SEQ ID NO:1, or a conservative variant ordegenerate sequence comprising one or more substitution, deletion,addition and/or insertion in the said nucleotide sequence, or a sequencehybridizable with the said sequence under moderate stringent condition,or a complementary sequence thereof, or a variant or derivative havingat least 95% homology and same or similar biological function to thesaid nucleotide sequence. In one embodiment of the present invention,the said promoter consists of the DNA sequence as shown in the DNAsequence of the positions 1-1373 of SEQ ID NO:1.

The gene or homologous gene of the present invention is able to bescreened from cDNA and genomic library by using apolynucleotide-specific oligonucleotide primer/probe such as the clonedOsNACx gene. Similarly, the OsNACx gene of the present invention and anyDNA fragment of interest or DNA fragment homologous to it can also beobtained from amplification of genome, mRNA and cDNA by using PCR(polymerase chain reaction) technology. A sequence comprising OsNACxgene can be isolated and obtained by using the above techniques, and atransgenic plant with enhanced tolerance to drought and salt stress canbe obtained by transforming a plant with the said sequence and anyexpression vector capable of inducing the expression of an exogenousgene in the plant.

For example, polymerase chain reaction can be used for amplifying thesequence from cDNA, wherein the said cDNA is prepared from the isolatedRNA. A sequence-specific primer for this amplification can be designedbased on the sequence as shown in SEQ ID NO:1, or can be purchased orsynthesized. Then, the PCR product can be separated by gelelectrophoresis and detected by methods well known by those skilledtechnicians in the art.

The term “polynucleotide molecule-specific oligonucleotide primer/probe”refers to an oligonucleotide sequence having at least 80%, preferably atleast 90%, more preferably at least 95% identity to the desiredpolynucleotide, or to an anti-sence oligonucleotide of a sequence thathas at least 80%, preferably 90%, more preferably 95% identity to thedesired polynucleotide.

The very useful oligonucleotide primer and/or probe in the presentinvention has at least 10-40 nucleotides. In one preferable embodiment,the oligonucleotide primer includes at least about 10 consecutivenucleotides of the said polynucleotide. Preferably, the oligonucleotideused in the present invention includes at least about 15 consecutivenucleotides of the said polynucleotide. The technologies based on PCRtest and hybridization in situ test are well known in the art.

Another aspect of the present invention relates to an expression vectorcomprising the said polynucleotide sequence and/or the said promotersequence. When the gene of the present invention is constructed into aplant expression vector, any strong promoter or inducible promoter canbe added before its starting nucleotide for transcription. When the geneof the present invention is constructed into a plant expression vector,enhancers can also be used, and these enhancer regions can be ATGinitiation codons, adjacent region initiation codons, etc., but must beidentical to the reading frame of the encoding sequence in order toensure the translation of whole sequence.

The expression vector carrying the polynucleotide sequence of the OsNACxgene of the present invention can be introduced into plant cells byconventional biological methods such as Ti plasmid, plant virus vector,direct DNA transformation, microinjection, electroporation and the like(Weissbach, 1998, Method for Plant Molecular Biology VIII, AcademyPress, New York, pp. 411-463; Geiserson and Corey, 1998, Plant MolecularBiology (2^(nd) Edition).

The plants of the present invention include but are not limited to:tomato, potato, tobacco, pepper, rice, corn, barley, wheat, Brassica,Arabidopsis, sunflower, soybean, poplar, and pine. Preferred plant isrice, also includes non-agronomic species which are useful in developingappropriate expression vectors such as tobacco, rapid cycling Brassicaspecies, and Arabidopsis thaliana.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing a heterologous gene andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques arewidely described in the art (See for example, Sambrook. et al (1989),Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., and Ausubel, F. M., et al (1989), Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.).

In general, these vectors comprise the polynucleotide sequence of theinvention (as described above) operably linked to a promoter and otherregulatory sequences (for example, enhancers, polyadenylation signals,etc.) required for expression in a plant.

Promoters used in the present invention include but are not limited toconstitutive promoters, tissue-, organ-, and developmentally-specificpromoters, and inducible promoters. Examples of promoters include butare not limited to: constitutive promoter 35S of cauliflower mosaicvirus; a wound-inducible promoter from tomato, leucine amino peptidase(“LAP”, Chao et al. (1999), Plant Physiol 120: 979-992); achemically-inducible promoter from tobacco, Pathogenesis-Related 1 (PR1)(induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acidS-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) orLAP promoter (both inducible with methyl jasmonate); a heat shockpromoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter(U.S. Pat. No. 5,057,422); and seed-specific promoters, such as thosefor seed storage proteins (for example, phaseolin, napin, oleosin, and apromoter for soybean beta conglycin (Beachy et al. (1985), EMBO J. 4:3047-3053)). All references cited herein are incorporated in theirentirety.

The expression cassettes may further comprise any sequences required forexpression of mRNA. Such sequences include, but are not limited totranscription terminators, enhancers such as introns, viral sequences,and sequences intended for the targeting of the gene product to specificorganelles and cell compartments.

A variety of transcriptional terminators are available for use inexpression of sequences using the promoters of the present invention.Transcriptional terminators are responsible for the termination oftranscription beyond the transcript and its correct polyadenylation.Appropriate transcriptional terminators and those which are known tofunction in plants include, but are not limited to, the CaMV35Sterminator, the tml terminator, the pea rbcS E9 terminator, and thenopaline and octopine synthase terminator (See for example, Odell et al.(1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56: 125; Guerineauet al. (1991) Mol. Gen. Genet., 262: 141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5: 141; Mogen et al. (1990) Plant Cell,2: 1261; Munroe et al (1990) Gene, 91: 151; Ballad et al. (1989) NucleicAcids Res. 17: 7891; Joshi et al. (1987) Nucleic Acid Res., 15: 9627).

In some embodiments of the present invention, the construct forexpression of the nucleic acid sequence of interest also includes aregulator such as a nuclear localization signal (Calderone et al. (1984)Cell 39: 499; Lassoer et al. (1991) Plant Molecular Biology 17: 229), aplant translational consensus sequence (Joshi (1987) Nucleic AcidsResearch 15: 6643), an intron (Luehrsen and Walbot (1991) Mol. Gen.Genet. 225:81), and the like, operably linked to the nucleic acidsequence encoding plant CPA-FAS.

In preparing the construct comprising a nucleic acid sequence encodingplant CPA-FAS, various DNA fragments can be manipulated, so as toprovide for the DNA sequences in the desired orientation (for example,sense or antisense) orientation and, as appropriate, in the desiredreading frame. For example, adapters or linkers can be employed to jointhe DNA fragments or other manipulations can be used to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resection, ligation, or the likeis preferably employed, where insertions, deletions or substitutions(for example, transitions and transversions) are involved.

Numerous transformation vectors are available for plant transformation.The selection of a vector for use will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers are preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing and Vierra (1982) Gene 19:259; Bevan et al. (1983) Nature 304: 184), the bar gene which confersresistance to the herbicide phosphinothricin (White et al. (1990) NuclAcids Res. 18: 1062; Spencer et al. (1990) Theor. Appl. Genet. 79: 625),the hph gene which confers resistance to the antibiotic hygromycin(Blochlinger and Diggelmann (1984) Mol. Cell. Biol. 4: 2929), and thedhfr gene, which confers resistance to methotrexate (Bourouis et al.(1983) EMBO J., 2: 1099).

In some preferred embodiments, the vector is adapted for use in anAgrobacterium mediated transfection process (See for example, U.S. Pat.Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all ofwhich are incorporated herein by reference). Construction of recombinantTi and Ri plasmids in general follows methods typically used with themore common bacterial vectors, such as pBR322. Additional use can bemade of accessory genetic elements sometimes found with the nativeplasmids and sometimes constructed from foreign sequences. These mayinclude but are not limited to structural genes for antibioticresistance as selection genes.

There are two systems of recombinant Ti and Ri plasmid vector systemsnow in use. The first system is called the “cointegrate” system. In thissystem, the shuttle vector containing the gene of interest is insertedby genetic recombination into a non-oncogenic Ti plasmid that containsboth the cis-acting and trans-acting elements required for planttransformation as, for example, in the pMLJI shuttle vector and thenon-oncogenic Ti plasmid pGV3850. The second system is called the“binary” system in which two plasmids are used; the gene of interest isinserted into a shuttle vector containing the cis-acting elementsrequired for plant transformation. The other necessary functions areprovided in trans by the non-oncogenic Ti plasmid as exemplified by thepBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some ofthese vectors are commercially available.

In other embodiments of the invention, the nucleic acid sequence ofinterest is targeted to a particular locus on the plant genome.Site-directed integration of the nucleic acid sequence of interest intothe plant cell genome may be achieved by, for example, homologousrecombination using Agrobacterium-derived sequences. Generally, plantcells are incubated with a strain of Agrobacterium which contains atargeting vector in which sequences that are homologous to a DNAsequence inside the target locus are flanked by Agrobacteriumtransfer-DNA (T-DNA) sequences, as previously described (U.S. Pat. No.5,501,967). One of skill in the art knows that homologous recombinationmay be achieved using targeting vectors which contain sequences that arehomologous to any part of the targeted plant gene, whether belonging tothe regulatory elements of the gene, or the coding regions of the gene.Homologous recombination may be achieved at any region of a plant geneso long as the nucleic acid sequence of regions flanking the site to betargeted is known.

In yet other embodiments, the nucleic acids of the present invention isutilized to construct vectors derived from plant (+) RNA viruses (forexample, brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus,cucumber mosaic virus, tomato mosaic virus, and combinations and hybridsthereof). Methods for the construction and use of such viruses aredescribed in U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794;5,977,438; and 5,866,785, all of which are incorporated herein byreference.

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant targeted for transformation. In someembodiments, the vector is maintained episomally. In other embodiments,the vector is integrated into the genome.

In some embodiments, direct transformation in the plastid genome is usedto introduce the vector into the plant cell (See for example, U.S. Pat.Nos. 5,451,513; 5,545,817; 5,545,818; PCT application WO95/16783). Thebasic technique for chloroplast transformation involves introducingregions of cloned plastid DNA flanking a selectable marker together withthe nucleic acid encoding the RNA sequences of interest into a suitabletarget tissue (for example, using biolistics or protoplasttransformation with calcium chloride or PEG). The 1 to 1.5 kb flankingregions, termed targeting sequences, facilitate homologous recombinationwith the plastid genome and thus allow the replacement or modificationof specific regions of the plastome. Initially, point mutations in thechloroplast 16S rRNA and rps12 genes conferring resistance tospectinomycin and/or streptomycin are utilized as selectable markers fortransformation (Svab et al (1990) PNAS, 87: 8526; Staub and Maliga,(1992) Plant Cell, 4: 39). The presence of cloning sites between thesemarkers allowed creation of a plastid targeting vector introduction offoreign DNA molecules (Staub and Maliga (1993) EMBO J., 12:601).Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab and Maliga (1993) PNAS, 90:913). Other selectable markers useful for plastid transformation areknown in the art and encompassed within the scope of the presentinvention. Plants homoplasmic for plastid genomes containing the twonucleic acid sequences separated by a promoter of the present inventionare obtained, and are preferentially capable of high expression of theRNAs encoded by the DNA molecule.

In other embodiments, vectors useful in the practice of the presentinvention are microinjected directly into plant cells by use ofmicropipettes to mechanically transfer the recombinant DNA (Crossway(1985) Mol. Gen. Genet, 202: 179). In still other embodiments, thevector is transferred into the plant cell by using polyethylene glycol(Krens et al. (1982) Nature, 296: 72; Crossway et al. (1986)BioTechniques, 4: 320); fusion of protoplasts with other entities,either minicells, cells, lysosomes or other fusible lipid-surfacedbodies (Fraley et al. (1982) Proc. Natl. Acad. Sci., USA, 79: 1859);protoplast transformation (EP 0 292 435); direct gene transfer(Paszkowski et al. (1984) EMBO J., 3: 2717; Hayashimoto et al (1990)Plant Physiol. 93: 857).

In still further embodiments, the vector may also be introduced into theplant cells by electroporation. (Fromm, et al. (1985) Pro. Natl Acad.Sci. USA 82: 5824; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602). In this technique, plant protoplasts are electroporated in thepresence of plasmids containing the gene construct. Electrical impulsesof high field strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form plant callus.

In yet other embodiments, the vector is introduced through ballisticparticle acceleration using devices (for example, available fromAgracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.) (Seefor example, U.S. Pat. No. 4,945,050; and McCabe et al. (1988)Biotechnology 6: 923). See also, Weissinger et al. (1988) Annual Rev.Genet. 22:421; Sanford et al. (1987) Particulate Science and Technology,5:27 (onion); Svab et al. (1990) Proc. Natl. Acad. Sci. USA, 87:8526(tobacco chloroplast); Christou et al. (1988) Plant Physiol., 87:671(soybean); McCabe et al. (1988) Bio/Technology 6:923 (soybean); Klein etal. (1988) Proc. Natl. Acad. Sci. USA, 85:4305 (maize); Klein et al.(1988) Bio/Technology, 6:559 (maize); Klein et al. (1988) PlantPhysiol., 91:4404 (maize); Fromm et al. (1990) Bio/Technology, 8: 833;and Gordon-Kamm et al. (1990) Plant Cell, 2:603 (maize); Koziel et al.(1993) Biotechnology, 11:194 (maize); Hill et al. (1995) Euphytica,85:119 and Koziel et al. (1996) Annals of the New York Academy ofSciences 792:164; Shimamoto et al. (1989) Nature 338:274 (rice);Christou et al. (1991) Biotechnology, 9:957 (rice); Datta et al. (1990)Bio/Technology 8:736 (rice); European Patent Application EP0,332,581(orchardgrass and other Pooideae); Vasil et al. (1993) Biotechnology,11:1553 (wheat); Weeks et al. (1993) Plant Physiol., 102:1077 (wheat);Wan et al. (1994) Plant Physiol. 104:37 (barley); Jahne et al. (1994)Theor. Appl. Genet. 89:525 (barley); Knudsen and Muller (1991) Planta,185:330 (barley); Umbeck et al. (1987) Bio/Technology 5:263 (cotton);Casas et al (1993) Proc. Natl. Acad. Sci. USA 90:11212 (sorghum); Somerset al. (1992) Bio/Technology 10:1589 (oat); Torbert et al. (1995) PlantCell Reports, 14:635 (oat); Weeks et al. (1993) Plant Physiol., 102:1077(wheat); Chang et al., WO 94/13822 (wheat); and Nehra et al. (1994) ThePlant Journal, 5: 285 (wheat).

In addition to direct transformation, in some embodiments, the vectorscomprising a nucleic acid sequence encoding a plant CPA-FAS of thepresent invention are transferred using Agrobacterium-mediatedtransformation (Hinchee et al. (1988) Biotechnology, 6:915; Ishida etal. (1996) Nature Biotechnology 14:745). Agrobacterium is arepresentative genus of the gram-negative Rhizomaceae. Its species areresponsible for plant tumors such as crown gall and hairy root disease.In the dedifferentiated tissue characteristic of the tumors, amino acidderivatives known as opines are produced and catabolized. The bacterialgenes responsible for expression of opines are a convenient source ofcontrol elements for chimeric expression cassettes. Heterologous geneticsequences (for example, nucleic acid sequences operatively linked to apromoter of the present invention), can be introduced into appropriateplant cells, by means of the Ti plasmid of Agrobacterium tumefaciens.The Ti plasmid is transmitted to plant cells on infection byAgrobacterium tumefaciens, and is stably integrated into the plantgenome (Schell (1987) Science, 237:1176). Species which are susceptibleinfection by Agrobacterium may be transformed in vitro. Alternatively,plants may be transformed in vivo, such as by transformation of a wholeplant by Agrobacteria infiltration of adult plants, as in a “floral dip”method (Bechtold N, Ellis J, Pelletier G (1993) Cr. Acad. Sci. III-Vie316:1194-1199).

Another aspect of the present invention relates to a host celltransformed or transfected by the above said expression vector. Thehosts that can be transformed by an expression vector comprising theOsNACx gene of the present invention include but are not limited totomato, potato, tobacco, pepper, rice, corn, barley, wheat, Brassica,Arabidopsis, sunflower, soybean, poplar, and pine, preferable rice.

Another aspect of the present invention relates to use of the above saidpolynucleotide sequence and/or promoter sequence for enhancing planttolerance to drought and/or salt stress. The plants of the presentinvention include but are not limited to tomato, potato, tobacco,pepper, rice, corn, barley, wheat, Brassica, Arabidopsis, sunflower,soybean, poplar, and pine, preferably rice.

In one embodiment of the present invention, the expression of the saidpromoter sequence is stress inducible expression, so that the saidpromoter is an inducible promoter. When the promoter fragment of thepresent invention and any gene of interest are simultaneously linked inan appropriate expression vector and used to transform a plant host, thestress inducible expression of the gene of interest enhances thetolerance of plant to stress.

BRIEF DESCRIPTION OF THE DRAWING

SEQ ID NO:1 in the sequence listing shows the DNA fragment sequencewhich is isolated and cloned in the present invention and comprises anOsNACx gene encoding region and a promoter region.

FIG. 1: The flow chart of isolation and identification of OsNACx gene.

FIG. 2: The result of homology comparison between OsNACx gene and NACtranscription factors using ClustalW software (a public software).

FIG. 3: The result of analysis on the integrate OsNACx gene sequence byGENSCAN gene structure forecasting software(http://genes.mit.edu/GENSCAN.html). In FIG. 3: Gn represents genenumber; Ex represents exon; Init represents gene initiation exon; Termrepresents gene termination exon; Prom represents fundamental promoter;PlyA represents PolyA; S represents DNA chain, wherein “+” representsthe DNA sequence chain which is inputted during the analysis process;Begin represents the initiation position of exon, promotor or polyA inthe inputted DNA sequence; End represents the termination position ofexon, promotor or polyA in the inputted DNA sequence; Len represents thesequence length (bp) of exon, promotor or polyA; Fr represents thetranslation reading frame (3 translation reading frames per DNAsequence); I/Ac represents the 3′ splice site score; Do/t represents the5′ splice site score; CodRg represents the translation region score; Prepresents the exon probability; Tscr represents the exon score.

FIG. 4: The OsNACx gene expression levels detected by Northernhybridization assay at different time points under stresses such asdrought, high-salinity, low temperature and ABA, etc.

FIG. 5: The expression situations of OsNACx gene in transgenic plants(the first is control, and the residues are transgenic and independenttransgenic plants).

FIG. 6: The growth of transgenic families with overexpression of OsNACxduring adult stage after 22 days of drought stress in field.

FIG. 7: The growth of transgenic families with overexpression of OsNACxduring seedling stage after 12 days of high salinity (200 mM) stress,wherein A represents the control, B represents the transgenic familyT40S19, C represents the transgenic family T40S24, D represents thetransgenic family T40S26, E represents the transgenic family T40S8, andF represents the transgenic family T40S25.

FIG. 8: Trans-activation activity of OsNACx gene which is testedaccording to the expression of LacZ reporter gene after a yeast cell istransformed by OsNACx gene.

FIG. 9: The subcellular localization and the self-promotor expression ofOsNACx gene in plant cells. FIG. 8A shows the expression of OsNACx-GFPin resistance callus detected by fluorescence microscope; FIG. 8B showsthe subcellular localization of OsNACx-GFP in callus cells, wherein (a)is the callus section dyed by fluorochrome propidium iodide, (b) is theexpression image of GFP under green fluorescence, (c) is the syntheticalresult of red and green fluorescences.

FIG. 10: The structural sketch of overexpression carrier PCAMBIA1301 ofthe present invention.

FIG. 11: The structural sketch of subcellular localization carrierPCAMBIA1381-EGFP of the present invention.

EXAMPLES

During the initial research period of the present invention, the cDNAclone 04I24 of the rice variety MingHui 63 (a rice variety which iswidely extended in China). The said cDNA is the cDNA fragment of OsNACxgene. The inventors of the present invention found that it is a newstress-associated regulation gene.

Specifically, (1) it was found by cDNA chip technique that theexpression amount of the said cDNA clone 04124 in the rice variety“Zhonghan No. 5” (a rice variety which was provided by ShanghaiAgriculture Academy of China and was publically used in China) increased3.5 times after drought stress treatment for 15 days. The results ofsequencing and analysis indicated that the product encoded thereby wasup to 64% homologous with OsNAC4 (FIG. 2). Due to the significantdifference of expression amount before and after drought treatment andthe functional characteristics of the clone, it is deemed that the geneof clone 04I24 participated in the expression of regulation gene underdrought stress; (2) according to the analysis of expression profile ofthe said gene under stress (see FIG. 4), it is found that the expressionof said gene increased significantly; and (3) the transgenic plant withoverexpression of the intact gene exhibited a significantly enhancedtolerance to drought and high-salinity (FIG. 6 and FIG. 7).

The above results show that OsNACx gene is a stress-associatedregulation gene and participates in the regulation of tolerance not onlyto drought but also high-salinity and coldness.

The present invention is further demonstrated with examples incombination with figures, and describes methods for isolating andcloning the DNA fragment comprising the whole encoding region of OsNACxgene and for verifying the function of OsNACx gene, based on the initialresearches of the present invention (the procedure of invention is shownin FIG. 1).

According to the following description and examples, a skilled in theart can determine the basic technical features of the present invention,and can make any change and modification to the present inventionwithout leaving the spirit and scope of the present invention in orderto adapt to various uses and conditions.

Example 1 Separation and Cloning OsNACx Gene and DNA FragmentsContaining OsNACx Gene

According to the analysis on expression profile of drought induciblegene of the rice variety “Zhonghan No. 5” (a rice variety which isprovided by Shanghai Agriculture Academy of China and publically used inChina), a strongly drought-inducible EST (expression sequence tag) (theexpression amount increased at least 3.5 times after drought stress) wasfound, and the analysis of its sequence indicated that this gene was amember of the transcription factor family NAC and was a sequence at the5′ end portion.

The corresponding cDNA clone J012130D02 was found out by searching theJapan rice span database (http://cdna01.dna.affrc.go.jp) and located atthe 3^(rd) chromosome BAC clone AC135594. According to the BAC clonesequence AC135594, its promotor region is predicted, and its primersPF(5-CAGAATTCAAAGCAACAGTGGAGAGAAAAC, sequence-specific primer plus jointEcoRI site) and FR(5-TAGGATCCCCGAGCCATCTCTTGAC, sequence specific primerplus joint BamHI) were designed. The sequence of 9781-12321 bp of BACclone AC135594 was amplified from the total DNAs of the rice variety“Zhonghan No. 5”, and the amplification product was the sequence 1-2540bp of the present invention (FIG. 3).

The specific steps comprised: extracting the total DNAs from the ricevariety “Zhonghan No. 5” (CTAB Extraction Method, Zhang, et al, geneticdiversity and differentiation of indica an japonica rice detected byRFLP analysis, 1992, Theor Appl Genet, 83, 495-499) as the templates foramplification, wherein the reaction conditions were: predegeneration at94° C. for 3 min; 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 3min, 30 circulations; and elongation at 72° C. for 5 min; linking theamplification product of PCR to pGEM-T vector (bought from Promega &co.); and screening and sequencing (ABI3730 sequencer) positive clonesto obtain the desired DNA fragment comprising OsNACx gene region and thepredicted self-promoter. This clone is designated as PGEM-NAC-PRO.

The total RNAs were extracted from the rice variety “Zhonghan No. 5”with TRIZOL reagent (Invitrogen & co.) after drought-stress treatment(the extraction was conducted according to the TRIZOL reagentspecification). By using a reverse transcriptase (Invitrogen & co.), thefirst-strand of cDNA was synthesized by its reverse transcription,wherein the reaction conditions were: 65° C. for 5 min, 42° C. for 50min, 70° C. for 10 min. By using the nest primersFF(5-TAGGTACCAGAAGCAAGCAAGAAGCGAT, plus joint KpnI) andFR(5-TAGGATCCCCGAGCCATCTCTTGAC, plus joint BamHI), it was amplified fromthe inverse transcription products, wherein the reaction conditionswere: predegeneration at 94° C. for 3 min; 94° C. for 30 sec, 55° C. for30 sec, 72° C. for 3 min, 30 circulations; elongation at 72° C. for 5min. The PCR products obtained by the amplification were linked topGEM-T vector (Promega & co.), and a positive clone was screened andsequenced to obtain the desired full-length gene. The said clone wasdesignated as PGEM-OsNACx.

Example 2 Detection of Inducible Expression of Rice Endogenous GeneOsNACx

The rice variety “Zhonghan No. 5” was used as material and treatedseparately with drought, coldness, high-salinity stress as well as ABAduring the 3 leaf stage. The drought treatment was conducted byimmersing the seedling root in 20% polyethylene glycol (PEG6000) for 0h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and then sampling. The coldness treatmentwas conducted by placing the seedling in a 4° C. growth chamber for 0 h,1 h, 8 h, 12 h, and then sampling. The high-salinity treatment wasconducted by immersing the seedling root in 200 mM/L NaCl solution for 0h, 4 h, 8 h, 16 h, and then sampling. The ABA treatment was conducted byimmersing the seedling root in 100 μM/L ABA solution for 0 h, 0.5 h, 3h, 6 h, 12 h, 24 h, and then sampling. The total RNAs of the leaves wereextracted (Trizol reagent from Invitrogen & co.), then subject to RNAmembrane transfer (according to the experimental methods of “MolecularCloning”, Science Press, Peking, 1999), and Northern hybrided wasconducted by using OsNACx as probe. The result showed that the OsNACxgene cloned in the present invention could be induced to express bydrought, high-salinity, coldness and ABA (FIG. 4), and was astress-associated transcription factor.

Example 3 Construction and Transformation of OsNACx Gene OverexpressionVector

According to the results of Example 2, the OsNACx gene of the presentinvention could be induced to express by drought, high-salinity,coldness and ABA. In order to further illustrate the function of thisgene, it was overexpressed in rice and verified by the phenotype oftransgenic plants.

The method comprised: enzymatically cleaving the positive clonepGEM-OsNACx plasmid of Example 1 with BamHI and KpnI, and recoveringexogenous fragments; in the meantime, enzymatically cleaving the genetictransformation vector pD35S1301 (which is reconstructed based on acommon vegetable genetic transformation vector pCAMBIA1301 fromAustralia CAMBIA Laboratory (Center for the Application of MolecularBiology to International Agriculture), carries double tobacco mosaicvirus promotor 35S with constitutive and over expressioncharacteristics, and is mediated by Agrobacterium) by the same way;after cleavage, exacting with chloroform:iso-pentanol (24:1), purifyingthe enzymatic cleavage product, conducting linkage reaction by using theenzymatic cleavage fragment comprising OsNACx gene and the enzymaticallycleaved pD35S1301 vector (see FIG. 10), transforming E. coliDH10β(Invitrogen & co.), and screening positive clone by enzymaticcleavage to obtain a transformed vector.

By using the rice genetic transformation system mediated byAgrobacterium, it is introduced into the rice variety “Zhong Hua 11” (arice variety which is provided by China Rice Institute and is publicallyused in China), and a transgenic plant is then obtained byprecultivation, infestation, co-culture, screening the callus withhygromycin resistance, differentiation, rooting, seedling training andtransplanting. Based on the method reported by Hiei, et al. (Efficienttransformation of rice, Oryza sativa L., mediated by Agrobacterium andsequence analysis of the boundaries of the T-DNA, 1994, Plant Journal6:271-282), the rice (Oryza sativa L.) mediated by Agrobacterium isoptimized by a process of mainly using the following steps and reagents.

(1) Abbreviations of Reagents and Solutions

The abbreviations of phytohormones used in culture mediums of thepresent invention are as follows: 6-BA (6-BenzylaminoPurine); CN(Carbenicillin); KT (Kinetin); NAA (Napthalene acetic acid); IAA(Indole-3-acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS(Acetosringone); CH (Casein Enzymatic Hydrolysate); HN (Hygromycin B);DMSO (Dimethyl Sulfoxide); N6max (N6 slather ingredient solution); N6mix(N6 micro constituent solution); Msmax (MS slather ingredient solution);Msmix (MS micro constituent solution)

(2) Formulas of Major Solutions 1) Preparation of N6 MacroelementsMother Liquor (Expressed on the Basis of 10× Concentration):

KNO₃ 28.3 g KH₂PO₄ 4.0 g (NH₄)₂SO₄ 4.63 g MgSO₄•7H₂O 1.85 g CaCl₂•2H₂O1.66 g

These compounds were dissolved one by one, and diluted to a meteredvolume of 1000 ml with distilled water at room temperature.

2) Preparation of N6 Microelements Mother Liquor (Expressed on the Basisof 100× Concentration):

KI 0.08 g H₃BO₃ 0.16 g MnSO₄•4H₂O 0.44 g ZnSO₄•7H₂O 0.15 g

These compounds were dissolved and diluted to a metered volume of 1000ml with distilled water at room temperature.

3) Preparation of Ferric Salt (Fe₂EDTA) Stock Solution (Expressed on theBasis of 100× Concentration):

800 ml double distilled water was prepared and heated to 70° C., then3.73 g Na₂EDTA.2H₂O was added, fully dissolved, kept in 70° C. waterbath for 2 h, diluted to a metered volume of 1000 ml with distilledwater, and stored at 4° C. for standby.

4) Preparation of Vitamins Stock Solution (Expressed on the Basis of100× Concentration):

Nicotinic acid 0.1 g VitaminB1 (Thiamine HCl) 0.1 g VitaminB6(Pyridoxine HCl) 0.1 g Glycine 0.2 g Inositol 10 g

Distilled water was added to a metered volume of 1000 ml, and stored at4° C. for standby.

5) Preparation of MS Macroelements Mother Liquor (Expressed on the Basisof 10× Concentration):

NH₄NO₃ 16.5 g KNO₃ 19.0 g KH₂PO₄ 1.7 g MgSO₄•7H₂O 3.7 g CaCl₂•2H₂O 4.4 g

These compounds were dissolved at room temperature and diluted withdistilled water to a metered volume of 1000 ml.

6) Preparation of MS Microelements Mother Liquor (Expressed on the Basisof 100× Concentration):

KI 0.083 g H₃BO₃ 0.62 g MnSO₄•4H₂O 0.86 g Na₂MoO₄•2H₂O 0.025 gCuSO₄•5H₂O 0.0025 g

These compounds were dissolved at room temperature and diluted withdistilled water to a metered volume of 1000 ml.

7) Preparation of 2,4-D Stock Solution (Expressed on the Basis of 1mg/ml):

100 mg 2,4-D was weighed and dissolved in 1 ml 1N potassium hydroxidefor 5 minutes, then 10 ml distilled water was added for completedissolution, the solution was diluted with distilled water to a meteredvolume of 100 ml and stored at room temperature.

8) Preparation of 6-BA Stock Solution (Expressed on the Basis of 1mg/ml):

100 mg 6-BA was weighed and dissolved in 1 ml 1N potassium hydroxide for5 minutes ago, then 10 ml distilled water was added for completedissolution, and the solution was diluted with distilled water to ametered volume of 100 ml and stored at room temperature.

9) Preparation of Naphthylacetic Acid (NAA) Stock Solution (Expressed onthe Basis of 1 mg/ml):

100 mg NAA was weighed and dissolved in 1 ml 1N potassium hydroxide for5 minutes, then 10 ml distilled water was added for completedissolution, and the solution was diluted with distilled water to ametered volume of 100 ml and stored at 4° C. for standby.

10) Preparation of Indoleacetic Acid (IAA) Stock Solution (Expressed onthe Basis of 1 mg/ml):

100 mg IAA was weighed and dissolved in 1 ml 1N potassium hydroxide for5 minutes, then 10 ml distilled water was added for completedissolution, and the solution was diluted with distilled water to ametered volume of 100 ml and stored at 4° C. in a triangular flask with300 ml distilled water and 2.78 g FeSO₄.7H₂O. 300 ml distilled water wasadded in another triangular flask for standby.

11) Preparation of Glucose Stock Solution (Expressed on the Basis of 0.5g/ml):

100 mg of glucose was weighed and dissolved and diluted with distilledwater to a metered volume of 250 ml, and stored at 4° C. aftersterilization.

12) Preparation of AS Stock Solution:

0.392 g of AS and 10 ml DMSO were packaged in a 1.5 ml centrifugal pipe,and then stored at 4° C. for standby.

13) Preparation of 1N Stock Solution:

5.6 g potassium hydroxid was weighed, dissolved and diluted withdistilled water to a metered volume of 100 ml, and stored at roomtemperature for standby.

(3) Culture Medium Formula for Genetic Transformation of Rice 1)Induction Culture Medium:

N6max mother liquor (10X) 100 ml N6mix mother liquor (100X) 10 mlFe²⁺EDTA stock solution (100X) 10 ml Vitamins stock solution (100X) 10ml 2,4-D stock solution 2.5 ml Proline 0.3 g CH 0.6 g Sucrose 30 gPhytagel 3 g

Distilled water was added to 900 ml, and pH value was adjusted to 5.9with 1N potassium hydroxide, then the medium was boiled and diluted to ametered volume of 1000 ml, and subpackaged in 50 ml triangular flasks(25 ml/flask), sealed and sterilized.

2) Secondary Culture Medium:

N6max mother liquor (10X) 100 ml N6mix mother liquor (100X) 10 mlFe²⁺EDTA stock solution (100X) 10 ml Vitamins stock solution (100X) 10ml 2,4-D stock solution 2.0 ml Proline 0.5 g CH 0.6 g Sucrose 30 gPhytagel 3 g

Distilled water was added to 900 ml, the pH value was adjusted to 5.9with 1N potassium hydroxide, then the medium was boiled and diluted to ametered volume of 1000 ml, subpackaged in 50 ml triangular flasks (25ml/flask), sealed and sterilized.

3) Pre-Cultured Medium:

N6max mother liquor (10X) 12.5 ml N6mix mother liquor (100X) 1.25 mlFe2+EDTA stock solution (100X) 2.5 ml Vitamins stock solution (100X) 2.5ml 2,4-D stock solution 0.75 ml CH 0.15 g Sucrose 5 g Agarose 1.75 g

Distilled water was added to 250 ml, and pH value was adjusted to 5.6with 1N potassium hydroxide, then the medium was sealed and sterilized.Before using, the medium was heated and melted, 5 ml glucose stocksolution and 250 μl AS stock solution were added, then the medium waspured into culture dishes (25 ml/dish). pH=5.6.

4) Cocultivation Medium:

N6max mother liquor (10X) 12.5 ml N6mix mother liquor (100X) 1.25 mlFe²⁺EDTA stock solution (100X) 2.5 ml Vitamins stock solution (100X) 2.5ml 2,4-D stock solution 0.75 ml CH 0.2 g Sucrose 5 g Agarose 1.75 g

Distilled water was added to 250 ml, pH value was adjusted to 5.6 with1N potassium hydroxide, and the medium was sealed and sterilized. Beforeusing, was heated and melted, 5 ml glucose stock solution and 250 μl ASstock solution were added, then the medium was pured into culture dishes(25 ml/dish).

5) Suspension Medium:

N6max mother liquor (10X) 5 ml N6mix mother liquor (100X) 0.5 mlFe²⁺EDTA stock solution (100X) 0.5 ml Vitamins stock solution (100X) 1ml 2,4-D stock solution 0.2 ml CH 0.08 g Sucrose 2 g

Distilled water was added to 100 ml, pH value was adjusted to 5.4, andthen the medium was subpackaged in two triangular flasks, sealed andsterilized. Before using, 1ml glucose stock solution and 100 μl AS stocksolution were added.

6) General Medium for Select Culture Medium:

N6max mother liquor (10X) 25 ml N6mix mother liquor (100X) 2.5 mlFe²⁺EDTA stock solution (100X) 2.5 ml Vitamins stock solution (100X) 2.5ml 2,4-D stock solution 0.625 ml CH 0.15 g Sucrose 7.5 g Agarose 1.75 g

Distilled water was added to 250 ml, pH value was adjusted to 6.0, andthen the medium was sealed and sterilized. Before using, the medium wasdissolved, 250 μl hygromycin (50 mg/ml) and 400 ppm carbenicillin (CN)were added, and then the medium was subpackaged in culture dishes (25ml/dish).

7) Pre-Differentiation Medium:

N6max mother liquor (10X) 25 ml N6mix mother liquor (100X) 2.5 mlFe2+EDTA stock solution (100X) 2.5 ml Vitamins stock solution (100X) 2.5ml 6-BA stock solution 0.5 ml KT stock solution 0.5 ml NAA stocksolution 50 μl IAA stock solution 50 μl CH 0.15 g Sucrose 7.5 g Agarose1.75 g

Distilled water was added to 250 ml, pH value was adjusted to 5.9 with1N potassium hydroxide, and then the medium was sealed and sterilized.Before using, 250 μl hygromycin (50 mg/ml) and 200 ppm carbenicillin(CN) were added, and then the medium was subpackaged in culture dishes(25 ml/dish).

8) Differentiation Medium:

N6max mother liquor (10X) 100 ml N6mix mother liquor (100X) 10 mlFe²⁺EDTA stock solution (100X) 10 ml Vitamins stock solution (100X) 10ml 6-BA stock solution 2 ml KT stock solution 2 ml NAA stock solution0.2 μl IAA stock solution 0.2 μl CH 1 g Sucrose 30 g Phytagel 3 g

Distilled water was added to 900 ml, pH value was adjusted to 6.0 with1N potassium hydroxide. The medium was then boiled and diluted to ametered volume of 1000 ml, and subpackaged in 50 ml triangular flasks(25 ml/flask), sealed and sterilized.

9) Rooting Culture Medium:

MSmax mother liquor (10X) 50 ml MSmix mother liquor (100X) 5 ml Fe2+EDTAstock solution (100X) 5 ml Vitamins stock solution (100X) 5 ml Sucrose30 g Phytagel 3 g

Distilled water was added to 900 ml, and pH value was adjusted to 5.8with 1N potassium hydroxide. The medium was then boiled and diluted to ametered volume of 1000 ml, and subpackaged in rooting tubes (25ml/tube), sealed and sterilized.

(4) Steps of Genetic Transformation Mediated by Agrobacterium (SpecificExamples of Using the Above-Mentioned Culture Mediums) 3.1 CallusInduction

-   (1) Mature rice seeds of “ZHONGHUA 11” were deshelled, then treated    with 70% alcohol for 1 minute and disinfected the surface of the    seeds with 0.15% HgCl₂ for 15 minutes in order;-   (2) The seeds were washed with sterilized water for 4-5 times;-   (3) The seeds were put on the above-mentioned induction medium;-   (4) The inoculated medium was placed in darkness and cultured for 4    weeks at 25±1° C. to obtained rice callus

3.2 Callus Subculture

The bright yellow, compact and relatively dry embryogenic callus wasselected, put onto the above-mentioned subculture medium, and culturedin darkness for 2 weeks at 25±1° C. to obtained rice callus.

3.3 Pre-Culture

The compact and relatively dry rice embryogenic callus was selected, putonto the above-mentioned pre-cultured medium, and cultured in darknessfor 2 weeks at 25±1° C.

3.4 Agrobacterium Culture

-   (1) Agrobacterium EHA105 (Invitrogen & co.) was pre-cultured on LA    culture medium (a publicly used medium) with corresponding    resistance at 28° C. for 48 h (2 days);-   (2) The Agrobacterium was transferred to the above-mentioned    suspension medium and cultured in a shaking table at 28° C. for 2-3    hours.

3.5 Agrobacterium Infection

-   (1) The pre-cultured callus was transferred into a sterilized    bottle;-   (2) The above said Agrobacterium was regulated to OD₆₀₀ 0.8-1.0;-   (3) The rice callus was immersed in the Agrobacterium suspension for    30 minute;-   (4) The callus of step (3) was transferred on sterilized filter    paper and dried, and then cultured onto the above-mentioned    cocultivation medium for 72 h (3 days) at 19-20° C.

3.6 Washing and Select Culture of Callus

-   (1) The rice callus was washed with sterilized water until no    agrobacrium was observed;-   (2) The rice callus of step (1) was immersed in sterilization water    containing 400 ppm carbenicillin (CN) for 30 minutes;-   (3) The callus of step (2) was transferred on sterilized filter    paper and dried;-   (4) The callus of step (3) was transferred on the above-mentioned    select medium and select-cultured for 2-3 times, 2 weeks for each    time (The above-mentioned select medium was heated and melted, then    cooled to about 60° C., and appropriate hygromycin and carbenicillin    were added. The screening concentration was 400 mg/l for hygromycin    and 400 mg/l for carbenicillin in the select medium for the first    culture, and was 250 mg/l for hygromycin and 250 mg/l for    carbenicillin in the select medium for the second and following    cultures.)

3.7 Differentiation

-   (1) The resistant rice callus obtained from the aforementioned    select culture medium was transferred to the pre-differentiation    medium, and cultured in darkness for 5-7 weeks;-   (2) The rice callus obtained from the pre-differentiation culture of    step 1 was transferred to the differentiation medium, and cultured    in lighting at 19-20° C. to obtain a transgenic rice plant.

3.8 Rooting

-   (1) The roots of transgenic rice plant generated during the    differentiation were cut off;-   (2) The plant was then transferred to rooting culture medium, and    cultured in lighting at 26° C. for 2-3 weeks.

3.9 Transplantation

The residual medium on roots of the transgenic rice plant was washedoff, the seedling with good roots was transferred in greenhouse, andmoisture was maintained in primal days.

The obtained transgenic rice plant was designated as T40SN (wherein T40Srepresents the vector number, N represents the transgenic rice variety“ZHONGHUA 11”). Finally, 36 independent transgenic rice plants wereobtained.

Example 4 Drought Screening of the OsNACx Gene Transgenic T1 Family inField

In order to verify whether the drought resistance of transgenic rice isrelated to OsNACx gene, the expression of OsNACx gene in some transgenicrice plants in the present invention was detected by Northernhybridization technology (FIG. 5 showed the Northern hybridizationresults, wherein the method was the same as Example 2). In the meantime,the screening of T1 generation plants with drought resistance of thepresent invention was conducted in field by the following steps. Theseeds of every family of T1 generation were immersed into an aqueoussolution of hygromycin (50 mg/ml), non-sprouting seeds were removed,other seeds were seeded in seedling bed, and rice seedlings in 5 leafstage were transplanted to a sandy land in an anti-drought greenhouse,where 20 individual plants of each transgenic family were planted in 2rows, and water supply was stopped in 3 weeks before heading stage. FIG.6 showed the drought screening results of transgenic families and thecontrol under drought stress for 22 days. The experiment results showedthat the drought resistance of transgenic plants was significantlyhigher than that of the control. The detection of relative water contentof two families were 83.9% and 85.2%, respectively, which indicated thatthe relative water content was not apparently different, so that theOsNACx gene was assuredly relative to drought resistance, and theoverexpression of the gene could improve the drought resistance oftransgenic plants. In order to further demonstrate that the gene of thepresent invention can improve the drought resistance of transgenicplants, the fruit ratio of transgenic families under drought stress wasanalyzed. The statistical data showed that the fruit ratio ofoverexpression transgenic plants was apparently higher than that of thecontrol, and the fruit ratio increased from 1.8% to 25% (as shown inTable 1), while the number of tillers and the weight of thousand grainshad not apparently difference, which proved in another aspect thatOsNACx gene was assuredly related to the enhancement of droughtresistance.

TABLE 1 Comparison of fruit ratio between transgenic families and thecontrol under drought stress Average Fruit Rice Number of Ratio ofsingle Standard Significant Difference Family Plants plant (%) DeviationCompare to Control CK 5 1.78 0.71 — T40S8 5 24.04 3.39 Significant(p <0.001) T40S24 5 23.97 3.46 Significant(p < 0.001) T40S25 5 22.97 3.14Significant(p < 0.001) T40S19 5 31.87 6.06 Significant(p < 0.001) T40S215 23.34 2.77 Significant(p < 0.001)

Example 5 High-Salinity Resistance Screening of OsNACx Gene TransgenicT1 Family in Seedling Stage

According to Example 4, it was proved that the drought resistance ofOsNACx transgenic plants in adult stage was apparently higher than thatof the control. In order to verify other stress-resistances of OsNACxtransgenic rice, it was treated with high-salinity stress in seedlingstage.

The specific method was as follows: transgenic overexpression plants offive families in T1 generation were selected, the T1 generation seedswere immersed into an aqueous solution of hygromycin (50 mg/ml),non-sprouting seeds were removed, and other seeds were seeded in smallround buckets (the soil used in the experiments was a mixture of southrice soil and sands in a ratio of 2:3; isometric water to equivalentsoil was added in each bucket; and the water naturally leaked out toensure the consistency of soil compactness). The experiment was repeatedfor 3 times. The healthy plants in 5 leaf stage was treated withhigh-salinity stress (200 mM NaCl solution) until all control plantsdied, and the survival rate of the plants were observed (individualplant with less than 20% green leaf area was usually difficult tosurvive and deemed to be dead). FIG. 7 showed the growth condition ofoverexpression transgenic family when the control plants all died underthe stress for 12 days. The table 2 showed the survival rate of everyoverexpression transgenic family when the control plants all died. Thesurvival rate of every transgenic family was usually higher than 80%.The result showed that OsNACx transgenic plants could improve theresistance of plants to high-salinity.

TABLE 2 Survival rate (%) of every transgenic family under high-salinitystress for 12 days Rice Repeated Survival Family Number SurvivalNumber/Total Number Rate TA0S25 3 21/24 23/27 24/29 85.1 ± 2.4 T40S24 320/26 24/28 23/27 82.6 ± 4.9 T40S21 3 18/21 21/24 19/26 82.1 ± 7.9 T40S83 25/27 24/28 26/30 88.3 ± 3.7 T40S19 3 21/23 25/27 24/29 88.9 ± 5.3

Example 6 Verification of the Transcription Activity of OsNACx Gene 3′End

Since the gene of the present invention was an inducible transcriptionfactor, it should possess function of transcriptional activation, andcould activate the downstream gene expression under stress in order togenerate resistance. In order to verify whether the OsNACx gene of thepresent invention has the function of transcriptional activation, orwhich region possesses the function, a yeast system was used in atrans-activation experiment in the present example.

Firstly, a series of OsNACx gene and partial deletion muton to yeastGAL4-DB fusion expression vector pDEST32 (Invitrogen & co.) wereconstructed, and used to transform yeast cell Y187 (CLONTHCH & co.). Inthe β-Galactosidase activity test, the expression of reporter gene LacZwas determined based on whether the yeast showed blue color. The resultsshowed that the OsNACx gene really possessed function of transcriptionalactivation, and the activation functional domain was located at the 3′end of the gene, and the amino acid sequence from 243 to 273 wasnecessary for the function of transcriptional activation of the presentgene. FIG. 8 showed the expression situation of reporter gene LacZ inyeast cell transformed by OsNACx gene or by partial deletion mutonthereof, wherein CK represents yeast cell Y187 transformed by emptyvector pDEST32; Full represents yeast cell Y187 transformed by pDEST32fused with full length OsNACx gene; Mut1 represents yeast cell Y187transformed by pDEST32 fused with 1-166AA fragment (NAM domain) ofOsNACx gene; Mut2 represents yeast cell Y187 transformed by pDEST32fused with 182-316AA fragment of OsNACx gene; Mut3 represents yeast cellY187 transformed by pDEST32 fused with 182-273AA fragment of OsNACxgene; Mut4 represents yeast cell Y187 transformed by pDEST32 fused with182-243AA fragment of OsNACx gene.

The specific method was as follows:

1. The full length OsNACx gene and partial deletion muton thereof werefused to the yeast expression vector pDEST32 (Invitrogen & co.).

According to the reading frame of pDEST32 carrier and the full lengthcDNA xx sequence, the following gene primers were designed (by usingsoftware primer 5.0), respectively:

DBF: 5-AGAAGCAAGCAAGAAGCGAT DBR: 5-CCGAGCCATCTCTTGAC DBM1R:5-TCCGACACAGCACCCAATCATC DBM3R: 5-TATCGTCGTAGCTCAGGTCCA DBM4R:5-CTTTCTTGGGCACCATCAT DBM5R: 5-ACGGGAAGGGGTCGTTGTCCA DBMF:5-CTGTACAACAAGAAGAACG

The joint attB1 (5-ggggacaagtttgtacaaaaaagcaggct) was then added to thefront-ends of primers DBF and DBMF; and the joint attB2 (5-ggggaccactttgtacaagaaagctgggt) was added to the front-ends of primers DBR, DBM1R,DBM3R, DBM4R and DBM5R. Under the reaction conditions: predegenerationat 94° C. for 3 min; 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 3min, 30 circulations; elongation at 72° C. for 5 min, the product of thecombination of primers DBF and DBMF was DBNACF (1-316AA), the product ofprimers DBF and DBM1R was Mut1 (1-182AA), the product of primers DBMFand DBR was Mut2 (182-316AA), the product of primers DBMF and DBM3R wasMut3 (182-273AA), and the product of primers DBMF and DBM4R was Mut4(182-243AA). The obtained PCR products were purified through PEG8000,and then subject to BP recombination reaction with intermediate vectorpDONR221 (Invitrogen & co.), wherein the reaction system were 5 ul, 200ng PCR product, 50 ng pDONR221, 2 ul 5XBP Clonase Reaction Buffer, and 2ul BP Clonase Mix. The E. coli DH10β (Invitrogen & co.) was transformedat 25° C. for 5 h, positive clones were screened, and then the genefragment carried by the desired positive clone plasmid was fused toyeast expression vector pDEST32 by LR recombination reaction, whereinthe steps comprised: E. coli DH10β (Invitrogen & co.) was transformedunder conditions of 100 ng positive plasmid of BP reaction, 50 ngpDEST32, 2 ul 5XLR Clonase Buffer, 2 ul LR Clonase Mix, at 25° C. forabout 5h, and positive clones were screened.

2. Preparation and transformation of yeast competent (CLONTECH, YeastProtocols Handbook) by lithium acetate (LiAc) method

1) Reagent and Formula

A. YPD nutrient solution:

20 g Difco peptone 10 g extractive of yeast 20 g glucosediluted to a metered volume of 1000 ml with distilled water, andsterilized for 15 minute.B. SD/Leu culture solution:

6.7 g  yeast nitrogenous base without amino acid 20 g agar powder 20 gglucose 0.69 g   Leu DO Supplement (CLONTECH & co.)diluted to a metered volume of 1000 ml with distilled water, andsterilized for 15 minute.C. 10TE buffer solution:0.1M Tris-HCl, 10 mM EDTA, pH 7.5, sterilized

D. 10LiAc:

1M lithium acetate, pH 7.5, sterilized

E. PEG/LiAc Solution:

Final concentration For preparation 10 ml solution PEG4000 40% 8 ml 50%PEG TE buffer solution 1x 1 ml 10x TE LiAc 1x 1 ml 10x LiAc

2) Procedure:

A. Yeast single colonies with diameter of 2-3 mm were scattered by 1 mlYPD solution, and then transferred to a triangular flask containing 10ml YPD medium.B. Cultured under the rotation of 250 rpm at 30° C. for 16-18 h, so thatOD600>1.5.C. 5 ml the above-mentioned yeast solution was transferred to anothertriangular flask containing 50 ml YPD medium and detected concentrationto get OD600=0.2-0.3.D. Cultured at 30° C. for 3 h (230 rpm), OD600=0.4-0.6 (if OD600<0.4,the culture maybe get in trouble).E. The yeast solution was transferred into a 50 ml centrifuge tube, andcentrifuged at 1000×g for 5 minute at room temperature.F. The supernatant was removed, the cells was suspended with sterilizeddouble distilled water again, and centrifuged at 1000×g for 5 minute atroom temperature.G. The supernatant was removed, the yeast cells were mixed homogenouslywith 1×TE/1×LiAc that was prepared in situ.H. 200 ng fusion plasmid DNA was transferred into a 1.5 ml centrifugetube, and 100 ul yeast competent cells were added and mixedhomogeneously, then 600 ul PEG/LiAc was added, centrifuged at highspeed, and cultured at 30° C. for 30 min (200 rpm).I. 70 ul DMSO (100%) was added, cantabily reversed for several times,placed in 42° C. water bath for 15 min, and then placed on ice for 2min.J. Centrifuged at 14000 rpm for 5 sec at room temperature, thesupernatant was removed, and the yeast cells were scattered with 1×TEbuffer.K. 100 ul transformed cells were coated on Leu/SD plate, inversioncultured in 30° C. incubator for 2-4 days, until clones appeared.3. Verification of transcription activity of OsNACx gene and partialdeletion muton thereof based on the expression of reporter gene LacZ inbeta-galactosidase experiment

1) Reagent and Formula

A. Z buffer solution

Na₂HPO₄•7H₂O 16.1 g/L NaH₂PO₄•H₂O 5.5 g/L KCl 0.75 g/L MgSO₄•7H₂O 0.246g/L

Regulated pH to 7.0, and sterilized.

B. X-gal stock solution (20 mg/ml)C. Z buffer solution/X-gal solution:100 ml Z buffer solution:0.27 ml β-mercaptoethanol1.67 ml X-gal stock solution

2) Procedure:

A. The transformed clone grew to 1-3 mm (30° C., 2-4 days).B. Round Watman filter paper with appropriate size was placed on 10 cmasepsis plate, and about 2.5-5 ml Z buffer/X-gal solution was added towet the filter paper, and bubble was avoided.C. Another clean asepsis filter paper was moved by forceps to place onthe plate with growing clone, and the filter paper was slightly pressedin order to adhere the clone to the filter paper.D. When the filter paper was wetted, it was opened with forceps, and thesurface with clone was upward, then the filter paper was placed intoliquid nitrogen for 10 sec, and thawed at room temperature in order tocrash the yeast cells.F. The filter paper which surface with clone was upward was carefullyplaced onto the previously wetted filter paper, and bubble was avoided.G. The filter paper was placed at 30° C. (30 min-8 hr), and theactivation function of the gene was judged according to the occurrenceof blue spot.

Example 7 Functional Verification of Osx Genic Promotor and it'sSubcellular Localization

In order to confirm the expression location of OsNACx gene in cell andthe activity of it's self promotor (1-1374 bp), the construction ofGFP-NLS (nuclear location signal) fusion protein was further performed,and the gene expression profile was determined according to theexpression of GFP. First, the published documents about Arabidopsisthaliana NAC gene (Miki Fujita, Kazuo Shinozaki et al., ADehydration-induced NAC protein, RD26, is involved in a novelABA-dependent stress-signaling pathway. Plant J (2004), 39, 863-876)were used. It was deduced that the nuclear location signal (NLS) of thegene may locate at 79-90AA or 116-182AA, and the subcellular location ofthe gene could be determined according to the expressive region of thissequence fused with GFP in cell.

The 1-1641 bp fragment (including first 1374 bp of ATG and gene 1-90AA,i.e, including promotor region and nuclear location signal) was fusedwith pCAMBIA1381-GFP vector. It was expected that 1-1374 bp of thefragment had already contained an intact promotor which could promotethe expression of gene, and that the 1-99AA sequence (including thenuclear location signal) of the gene was included so that the expressivecondition of the gene in cells could be determined according to theexpressive location of GFP. The pCAMBIA1381-EGFP vector wasreconstructed (see FIG. 11) based on pCAMBIA1381 (a plant genetictranscription vector commonly used in the world), wherein the carriedGUS gene was replaced with GFP gene, and no promoter was before the GFPgene. The pCAMBIA1381 vector was from Australia CAMBIA Laboratory(Center for the Application of Molecular Biology to InternationalAgriculture).

The specific method for the construction of vector for the fused genewas as follows. The primers PF (5-cagaattcaaagcaacagtggagagaaaac, addedwith joint EcoRI site) and PR (5-caaagcttgcgtgacccattaggatactt, addedwith joint HindIII) were designed, then the total DNAs of rice variety“Zhonghan No. 5” or the vector PGEM-NAC-PRO constructed in Example 1were used as template, and the amplification program (predegeneration at94° C. for 3 min; 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 3min, 30 circulations; elongation at 72° C. for 5 min) were employed. Theamplification product was enzymatically cleaved by EcoRI and HindIII andlinked to the vector pCAMBIA1381-EGFP that was enzymatically cleaved byEcoRI and HindIII as well. The rice callus was transformed with thefusion vector p1381-GFP-promoter-NLS using Agrobacterium-mediated genetransformation system (see detailedly in Example 3), the callus withhygromycin resistance was obtained (see Example 3), and the expressionof GFP was observed under fluorescence microscope (see FIG. 9A). Theresult showed that 1-1374 bp of the sequence had already contained theintact promoter, and it could promote expression of the gene.

In order to localize the gene in cell, the resistance callus was slicedand observed under confocal microscope to determine the intracellularexpression condition of GFP. FIG. 9B showed that GFP was expressed onlyin nuclei under the observation of confocal microscope, which indicatedthat the sequence 1-99AA had already included NLS so that GFP could belocalized in nuclei, i.e., the OsNACx protein was localized in nuclei.This example proved that the sequence 1-1374 bp in the present inventionincluded intact promotor, and it could induce the expression of thegene. In addition, it was speculated that the 79-90AA of OsNACx was NLS,and thus the OsNACx protein was localized in cell nucleus.

1. An isolated polynucleotide capable of giving a plant tolerance to drought and/or salt stress, which comprises a nucleotide sequence as shown in SEQ ID NO:1, or a conservative variant or degenerate sequence comprising one or more substitutions, deletions, additions and/or insertions in the said nucleotide sequence, or a sequence hybridizable with the said sequence under moderate stringent condition, or a complementary sequence thereof, or a variant or derivative having at least 95% homology and same or similar biological function to the said nucleotide sequence.
 2. The polynucleotide according to claim 1, which consists of the DNA sequence as shown in SEQ ID NO:1.
 3. The polynucleotide according to claim 1, which consists of the DNA sequence as shown in the positions 1374-2453 of SEQ ID NO:1.
 4. A promoter capable of giving a plant tolerance to drought and/or salt stress, which comprises a nucleotide sequence as shown in the DNA sequence of the positions 1-1373 of SEQ ID NO:1, or a conservative variant or degenerate sequence comprising one or more substitutions, deletions, additions and/or insertions into the said nucleotide sequence, or a sequence hybridizable with the said sequence under moderate stringent condition, or a complementary sequence thereof, or a variant or derivative having at least 95% homology and same or similar biological function to the said nucleotide sequence.
 5. The promoter according to claim 4, which consists of the DNA sequence as shown in the DNA sequence of the positions 1-1373 of SEQ ID NO:1.
 6. The polynucleotide according to claim 1 or the promoter according to claim 4, wherein the said plant is selected from the group consisting of rice, tomato, potato, tobacco, pepper, corn, barley, wheat, Brassica, Arabidopsis, sunflower, soybean, poplar and pine, preferably rice.
 7. An expression vector comprising the polynucleotide according to claim 1 and/or the promoter of claim
 4. 8. A host cell transformed or transfected by the expression vector according to claim
 7. 9. Use of the polynucleotide according to claim 1 and/or the promoter according to claim 4 for increasing tolerance to drought and/or salt stress in a plant.
 10. The use according to claim 9, wherein the said plant is selected from the group consisting of rice, tomato, potato, tobacco, pepper, corn, barley, wheat, Brassica, Arabidopsis, sunflower, soybean, poplar and pine, preferably rice. 